The depressed calcium cycling in animal models of heart failure and human failing hearts has been suggested to reflect, at least in part, the impaired calcium sequestration by the sarcoplasmic reticulum (SR). Calcium sequestration by the SR is mediated by a Ca2+-transport ATPase (SERCA2), whose activity is reversibly regulated by the phosphorylation status of phospholamban (PLN). Dephosphorylated PLN is an inhibitor of the Ca-affinity of SERCA2 and cardiac contractility, while phosphorylation of PLN relieves the inhibitory effects. The levels of PLN have been shown to be a major determinant of basal contractility and the heart's responses to 13-agonists. In human heart failure, the levels of PLN relative to SERCA2 are increased, resulting in increased inhibition of the Ca-pump's Ca-affinity and prolonged relaxation. In addition, the phosphorylation status of PLN is decreased, resulting in increased inhibitory function by PLN and further depression of SR Ca-cycling. The decreased PLN phosphorylation reflects increases in the PLN phosphatase 1 activity, partially due to dephosphorylation and attenuation of the inhibitor 1 (I-1) function. Thus, these two major Ca-regulatory proteins become our focus of attention in heart failure and it is important to determine whether genetic variations, such as point mutations, may occur in the PLN and I-1 human genes, which may alter their activities and modify the clinical course of the disease. Indeed, we have recently identified two human PLN mutations that led to dilated cardiomyopathy and heart failure at a young age. We propose here to continue our studies on identification of additional mutations in the PLN as well as the 1-1 genes and determine their functional significance and pathophysiological relevance, utilizing expression systems and genetically altered mice. In parallel, the subjects with the identified mutation will be closely followed to assess their cardiac function, exercise tolerance and force-interval relations (mechanical and relaxation restitution). Our findings will determine whether correlations may be established between a specific mutation in PLN or 1-1 and a specific clinical parameter or the time course of heart failure. Furthermore, these studies will provide valuable insights into the mechanisms by which specific genetic variants alter SR Ca-handling and function in heart failure.